RNA and DNA Extractions for Brown/Barott Pdam Project, November 11, 2022

Protocol Link

Extraction of 14 samples from Pocillopora damicornis (Pdam) from Heron Island in RNAlater March 2021 (Kristen Brown, Postdoc in Katie Barott lab)

Samples

Notes

• Continuting processing the Barott and Brown Pdam samples using the protocol detailed on 10/27/22.

- Had much higher qubit values here because I started putting two of the tiny fragments into the bead beat tubes instead of one. Mostly this meant I only had to bead beat most samples for 1 minute instead of 2 to get a dark colored solution and resulted in great RNA! But I think the previous samples are great too, just slightly less in concentration.

• See bead-beating notes below with Qubit values.

Qubit Results

• Used Broad range dsDNA and RNA Qubit Protocol
• All samples read twice, standard only read once

DNA Standards: 191.69 (S1) & 22436.66 (S2)

Sample #	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
RS6A	24.8	24.6	24.7
RS8C	51.4	50	50.7
RS9A	22.2	22.2	22.2
RS11D	36.4	35.6	36
RS12A	81	80.2	80.6
RS13C	41.2	40.2	40.7
RS14B	29.4	28	28.7
RF16A	47.2	46.8	47
RF17B	27	26.6	26.8
RF18D	24.4	24.2	24.3
RF19C	18.2	17.8	18
RF20C	56.4	55.6	56
RF23A	41.6	41.8	41.7
RF23C	18.6	18.2	18.4

RNA Standards: 422.67 (S1) & 9120.78 (S2)

Sample #	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG	Bead Beat Time (mins)
RS6A	36.2	36	36.1	1.5
RS8C	51.6	51.4	51.5	1
RS9A	22.2	22	22.1	1.5
RS11D	35	34.8	34.9	1.5
RS12A	58.2	57	57.6	1
RS13C	47	46	46.5	2
RS14B	37.6	38	37.8	2
RF16A	43.2	43.2	43.2	3
RF17B	34.2	34.6	34.4	2
RF18D	36.6	37.2	36.9	2
RF19C	28.8	28.6	28.7	2
RF20C	42.8	42.6	42.7	1
RF23A	47.6	47.2	47.4	1
RF23C	35.6	35.4	35.5	1.5

DNA and RNA Quality Check: Gel, using Kristina’s gel protocol at the bottom of this page.

And yes, we ran out of ladder. I think we have more but I could not find it.

Next steps

All done with these samples! Will check in with Hollie to see next steps for the extracted RNA and DNA. We could maybe redo some of the lower concentration extractions and add more tissue into the bead-beating steps for those.

I did re-QC the following five samples that had originally “nd” values for RNA quantity when measured using the RNA Broad Range Qubit kit. I thawed the RNA on ice and measured their RNA quantity using the Qubit RNA High Sensitivity kit, which uses different reagents and standards but the exact same protocol as the BR kit..

Sample #	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG
RF25A	10.5	10.3	10.4
RS14C	6.48	6.48	6.48
RS15D	6.84	7.04	6.94
RF14B	12.7	12.6	12.65
RF15D	11.6	11.1	11.35

All of them look okay, but we could possibly redo the extractions with more tissue at the bead-beat step, especially for those with concentrations below 10.